Multiphoton imaging of thoughts of food during natural and induced hunger states. To map the functions of the brain, neuroscientists often focus on brain responses to sensory stimuli and motor actions during engaging task conditions. However, remarkably little is known about the neural underpinnings of far more common and metabolically costly processes that occur during the 'resting state', such as mental imagery, cravings, and other processes that can powerfully influence future actions. For example, hunger can selectively induce imagery of food-associated cues (e.g. a candy bar wrapper), leading to powerful cravings and consumption of unhealthy foods. Human neuroimaging studies have taught us that imagery of objects results in increases in brain activity in the same lateral corticl areas that are activated by actual viewing of these objects. Electrophysiology studies in rat cortex have provided evidence for cellular correlates of mental imagery. Based on these studies in humans and rats, we hypothesize that hunger signals elicit a higher incidence of food-cue replay, defined as the endogenous reactivation of a specific set of cortical neurons previously found to respond selectively to a food-associated sensory cue. Food-cue replay may be critical for anticipating the reappearance of rewarding cues. Nevertheless, the neural basis for food-cue replay remains poorly understood because previous electrophysiological efforts typically recorded from only tens of unidentified neurons across short timescales of minutes to hours. We will overcome these limitations by measuring food-cue replay using novel two-photon calcium imaging techniques in awake, head-fixed mice. These methods will enable recording across many hours and days from the same population of over 1000 identified cortical cell bodies across all six layers of lateral cortex, and from large numbers of amygdalar axonal inputs to cortex that may bias cortex towards food-cue-specific replay. First, we will assess whether this genetic mouse model of food-cue replay captures the key features of hunger-driven thoughts of food in humans, including increased incidence during states of hunger, and subsequent augmentation of perceptual detection of sensory food cues and propensity to eat. We will then investigate the motivational drivers of food-cue replay by combining cortical imaging with optogenetic tools for monitoring and reversible manipulation of neural activity in hypothalamic AgRP neurons known to drive intense food seeking in mice. These experiments will begin to reveal the mechanisms by which a specific motivation - hunger - can selectively induce neural replay of food cues. Establishment of this novel mouse model will enable development and testing of clinical therapies that selectively decrease inappropriate thoughts about unhealthy foods and other addictive substances, thereby reducing the incidence of cravings that perpetuate food and drug addiction. More generally, this work will establish a powerful platform for basic and clinical research into the dark matter of neuroscience: endogenous cortical brain activity.